The bcl-2 translocation is the most common translocation in human cancer. Seventy-five percent of the bcl-2 translocations occur in a 150 bp region called the major breakpoint region (Mbr). Yet it has been unclear why this region of the genome is so focally fragile. The basis for other fragile sites in the genome is also not known. Recently, we determined that the bcl-2 Mbr adopts a structure which deviates from the normal B-form Watson-Crick duplex. We also determined that this non-B structure is cleaved by the RAG complex. The translocation arises because two of the four DMA ends from the V(D)J recombination process become rejoined with the two broken DNA ends at the bcl-2 Mbr. Many questions remain. First, how does the RAG complex bind to the various ends during the translocation process, and how does it cleave them? Aim 1 describes studies directed at understanding the RAG binding and cleavage of the Mbr in the presence and absence of the 12- and 23-signals. Second, how are the DNA ends put back together? Nonhomologous DNA end joining is the likely pathway. Aim 2 draws upon reagents created during the previous cycles of this project to test whether DNA ligase IV, the most critical component of this joining pathway, is required. Third, what is the precise structure of the bcl-2 Mbr region? It is very difficult to determine the precise structure of regions that deviate from the simple double-stranded form. However, we have made good progress on this, and Aim 3 proposes to use many different methods to characterize the precise structure of the non-B conformation. Preliminary data suggests that the conformation may be a triplex for at least part of the Mbr region. Fourth, can the 150 bp bcl-2 Mbr be positioned elsewhere in the human or murine genome to re-create the chromosomal fragility? Also, will the Mbr create fragile sites even in tissues that do not express the RAG complex. Aim 4 describes sensitive assays to test this in the human genome and in a mouse model. In the mouse model, the human Mbr is positioned at various locations in transgenic mice. Aims 1 through 4 will advance our understanding of chromosomal fragile sites in a general way because the DNA structural principles are likely to apply to other chromosomal fragile sites.